RT-PCR (Reverse Transcription Polymerase Chain Reaction) is a method that combines Reverse Transcription (RT) with PCR (Polymerase Chain Reaction). By combining cDNA synthesis with RNA as the template, e.g. RT, with PCR amplification, RT-PCR provides a rapid and sensitive method for the analysis of gene expression. RT-PCR detection or quantitative analysis of RNA is more sensitive and much easier to operate than other RNA analysis techniques such as Northern blotting, RNase protection analysis, hybridization in situ, and S1 nuclease analysis, and it has developed into a powerful means for detecting and analyzing gene expression levels and pathogens. However, in the application of RT-PCR, we have also encountered some problems, such as: high requirement for the purity of RNA sample—it requires not only the removal of non-nucleic acid substance such as protein, but also the removal of DNA in order to avoid interference of DNA; because it involves multiple operating procedures and long period of operation, it needs special care to prevent RNA degradation in the operation; since the two enzymatic reactions (RT and PCR) have different optimal reaction conditions, it is not easy to find an optimal balance point; all these issues lead to complicated procedure, high operating requirements and render it susceptible to various inhibitory substances and the trouble of non-specific amplification and false positive result. Meanwhile, the cost of reverse transcriptase has been always high and it is difficult to lower the implementation cost of this process. To sum up, the method has troublesome to carry out and the cost is high, resulting in difficulty in its clinical popularity.
Template-Ready PCR (TRPCR) is a RNA PCR detection method recently reported by the applicant (CN102864233A). In this method, an anchored probe is used to specifically bind with the target RNA, and another template probe that binds with target RNA is adsorbed and immobilized in the reaction tube, after repeated washing steps, unbound RNA and other substances are removed and a PCR reaction system is added for PCR amplification of the template probe, and the result can reflect the situation of presence of target RNA. Compared to traditional RT-PCR method, this process does not need purification and extraction of RNA or reverse transcription reaction, on the other hand, it decreases the previously three hours of PCR pre-treatment process to be 50 minutes. So it is simple, rapid and easy to operate, with good detection sensitivity and specificity for RNA, and is suitable for detecting RNA obtained from the lysate of various sources. While in practical application, the inventor has found that multiple washing steps of TRPCR are still complicated, and there is high requirement for temperature control of binding between template probe and target RNA and the procedure is easily influenced by the environmental temperature; if the room temperature is too low, then non-specific binding occurs, resulting in false positive result and bringing unstable and inconsistent factor to the result. Therefore the inventor has redesigned the TRPCR process and made major innovative improvement thereto by introducing a double-stranded DNA-specific digestion reaction.